This project analyzes the ability of a Saccharomyces cerevisiae vector containing the transgene encoding CEA (yeast-CEA) to activate human dendritic cells (DCs) and stimulate CEA-specific T-cell responses. We demonstrate for the first time that treatment with yeast-CEA can activate human DCs, resulting in increases in surface expression of CD80, CD83, CD54, CD58, and MHC class II, and increased production by DCs of IL-12p70, TNF-alpha, IFN-gamma, IL-8, IL-2, IL-13, IL-10, and IL-1beta. We also show that human DCs treated with yeast-CEA can activate CEA-specific T-cell lines and can act as antigen-presenting cells (APCs) to generate CEA-specific T-cell lines capable of lysing CEA+ human tumor cells. Gene expression profiles of human DCs treated with yeast-CEA show increased expression of numerous genes involved in the production of chemokines and cytokines and their receptors, and genes related to antigen uptake, antigen presentation, and signal transduction. Enhanced functionality of CD4+CD25highFoxP3+ regulatory T cells in the peripheral blood of patients with prostate cancer. CD4+CD25highFoxP3+ regulatory T cells (Treg) have been shown to inhibit the activation and function of T cells that participate in antigen-specific immune responses. Higher levels of Tregs have been reported in the peripheral blood of patients with several types of tumors. In this study, we investigated the number and functionality of CD4+CD25highFoxP3+ Tregs in patients with prostate cancer (PCa), and their potential role in inhibiting antitumor immune responses. Levels of Tregs in the peripheral blood of healthy donors and patients with biochemically progressive, localized, and metastatic PCa were each measured by flow cytometry. The functional activity of Tregs was determined by their ability to suppress the proliferation of CD4+CD25- T cells. Data were analyzed using Wilcoxon rank sum test and unpaired Students t test. Although levels of Tregs in the peripheral blood of patients with PCa were not significantly higher than those in healthy donors, Tregs in patients with PCa had significantly greater suppressive functionality than Tregs from healthy donors (P &lt;0.05). Additionally, there was a direct correlation between the serum levels of prostaglandin E2 and Treg functionality in patients with localized PCa, using Pearsons product-moment correlation coefficient (R). These findings further show the potential importance of Tregs in modifying immune responses in patients with PCa. Although longer studies are necessary to confirm these findings, these studies also show for the first time the differences in Treg populations in patients with various stages of PCa, and thus, provide a basis for determining which PCa patient populations are best suited for immunotherapy trials involving the inhibition of Tregs. We compared the effects of yeast-treated human dendritic cells (DCs) with CD40L-matured human DCs for the induction of effector cells and the number and functionality of CD4+CD25+CD127-FoxP3+ regulatory T cells (Tregs). DCs were treated with yeast or CD40L and cocultured with isolated autologous CD4+ T cells. CD4+CD25+CD127- T cells isolated from the coculture of CD4+ T cells plus yeast-treated DCs (yeast coculture) had a lower expression of FoxP3 and decreased suppressive function compared to CD4+CD25+CD127- T cells isolated from the coculture of CD4+ T cells plus CD40L-treated DCs (CD40L coculture). Also, compared to the CD40L coculture, the yeast coculture showed increases in the ratio of CD4+CD25+ activated T cells to Tregs and in the production of Th1-related cytokines (IL-2, TNF-alpha, IFN-gamma) and IL-6. In addition, yeast-treated DCs used as antigen-presenting cells (APCs) incubated with the tumor antigen CEA enhanced the proliferation of CEA-specific CD4+ T cells compared to the use of CD40L-matured DCs used as APCs. This is the first study to report on the role of yeast-treated/matured human DCs in reducing Treg frequency and functionality and in enhancing effector to Treg ratios. These results provide an additional rationale for the use of yeast as a vector in cancer vaccines. We have previously shown that the suppressive function of regulatory T cells (Tregs) from peripheral blood mononuclear cells (PBMCs) is enhanced in patients with prostate cancer when compared with healthy individuals. Two phase II studies using the PSA-TRICOM vaccine in patients with metastatic castration-resistant prostate cancer (mCRPC) showed evidence of patient benefit in terms of enhanced survival. The Halabi nomogram has been used to predict survival (HPS) of patients with mCRPC treated with conventional chemotherapy or second-line hormonal therapy. Tregs from PBMCs of patients (n = 23) with mCRPC were obtained pre- and post-three monthly vaccinations, and analyzed for number, phenotype, and suppressive function. Changes post- versus pre-vaccination in these parameters were compared with 3-year survival and HPS. No differences in Treg numbers were observed post- versus prevaccination. Trends (P = 0.029) were observed between overall survival (OS) and a decrease in Treg suppressive function post- versus pre-vaccination. Trends were also observed in analyzing effector:Treg (CD4+CD25+CD127-FoxP3+CTLA4+) ratio post- versus pre-vaccination with OS versus HPS. These data provide preliminary evidence for a possible association between improved OS and a decrease in Treg function when PBMCs are analyzed after three monthly vaccinations. Patients with an OS greater than HPS were more likely to have decreased Treg function following vaccine. Larger studies to confirm and extend these findings are warranted.